高盐摄入通过SGLT2调控血糖稳态的机制

发布时间：2018-05-19 09:34

本文选题：盐 + PPARδ　；参考：《第三军医大学》2017年博士论文

【摘要】：背景和目的:糖尿病患者的高血压管理是降低心血管死亡率和发病率的关键。长期以来,高血压的饮食调节方案重点关注在食盐摄入上。因此各大指南推荐糖尿病合并高血压患者限制盐的摄入。然而,限盐的潜在效益仍存有争议,一些证据显示,限盐会增加糖尿病合并高血压患者的心血管及全因死亡率,且高糖饮食对升高血压的影响远远大于高盐摄入。然而,关于盐摄入与血糖稳态之间的紧密联系的研究仍缺乏,人们对此知之甚少。人体肾脏可调节体液和电解质的平衡,同时对葡萄糖的过滤和重吸收起着至关重要的作用。其中,分布在肾脏皮质近端小管上皮细胞管腔侧的钠-葡萄糖协同转运蛋白(SGLTs),具有调节钠、糖重吸收的功能,能以较高效率以1:1协同转运Na+和葡萄糖。在高糖状态下,SGLT2的活性增加,对葡萄糖和钠的重吸收增强。而在糖尿病大鼠动物模型中,SGLT2突变可防止高糖诱导的多器官功能损害。选择性SGLT2抑制剂可改善糖尿病患者的高血糖,并有呈现出轻微的降压作用。近10年来,过氧化物酶体增殖物激活受体(peroxisome proliferator-activated receptor,PPAR)已经成为心血管代谢疾病的新型有效治疗靶点。人类肾脏表达不同的三种PPAR亚型,PPARα,PPARγ和PPARδ,在机体的糖脂代谢中发挥了重要作用。现今糖尿病治疗用噻唑烷二酮类(TZD)药物,就是通过激活脂肪组织PPARγ改善胰岛素抵抗,从而发挥改善血糖的作用。然而,研究表明TZD类药物可能导致水钠潴留及心衰,大大限制了其临床应用。PPARδ在几乎所有的细胞与组织中均存在表达,被认为参与了许多慢性病的发病过程,包括高血压、糖尿病、粥样硬化以及肥胖等,其特异性激动剂或基因表达上调均可有效缓解肥胖伴糖尿病啮齿类动物的高血脂、血糖和胰岛素抵抗。在糖尿病大鼠中,特异性激动PPARδ表达后表现出肾脏保护作用。此外,PPARδ激动剂增加的脂肪脂联素的表达,可在心血管疾病中发挥有益作用。然而,目前尚不清楚PPARδ是否通过调节脂联素表达参与了肾脏对钠的处置以及对葡萄糖的转运。脂联素是脂肪组织尤其是内脏脂肪组织分泌最丰富的蛋白质产物。脂联素通过其特异性受体,AdipoR1和AdipoR2,在多种组织和细胞中发挥其生理功能。已有研究发现PPARs在脂联素的表达调控中发挥了重要作用。应用PPARδ激动剂可显著升高内脏脂肪脂联素的表达水平,从而发挥改善心血管代谢紊乱的综合效应。血清脂联素水平在多种代谢性心血管疾病中降低,同时低脂联素水平可能是胰岛素抵抗发生、发展的重要原因。摄盐量对脂联素分泌有重要作用,低盐干预可显著上调脂肪组织脂联素分泌,同时改善胰岛素抵抗以及全身炎症反应。但脂联素如何参与盐的代谢尚不明确。在本研究中,我们提出PPARδ通过调节脂联素表达参与了肾小管SGLT2对钠的转运和葡萄糖的重吸收过程,干预该环节中的一个或多个关键靶点可维持或恢复钠和葡萄糖稳态的科学假设,并开展了一系列的细胞、动物实验及临床验证,为了验证上述假设,本研究分为两部分进行:1、验证高盐摄入影响血糖稳态及盐排泄,侧重观察PPARδ介导的脂联素分泌对SGLT2产生作用及其对尿钠及尿糖排泄的影响及其机制。2、在糖尿病动物模型中观察高盐摄入对SGLT2作用的影响及其机制,并在临床糖尿病患者中验证,结果表明脂肪PPARδ在脂联素介导的肾脏SGLT2调节钠糖平衡中发挥关键作用。材料与方法:本研究包括两部分:1.验证高盐对尿钠排泄和葡萄糖稳态的影响,研究高盐致脂肪PPARδ激活后由脂联素介导的促进小鼠肾脏尿钠和葡萄糖排泄的分子机制,同时在小鼠模型中测试SGLT2的急性功能改变。2.在第一部分实验基础上,在db/db糖尿病动物模型中研究高盐摄入对SGLT2表达及功能的影响,并在临床糖尿病患者中分析血糖、脂联素、尿钠三者的关系。1.3T3-L1前脂肪细胞以及C57BL/6小鼠原代脂肪细胞的分组培养,免疫印迹检测PPAR各亚型的表达,并设立氯化钠和甘露醇梯度的渗透压浓度,作为对照观察PPARδ的表达变化。2.脂肪特异敲除PPARδ小鼠(Fabp4-PPARδflox/flox)模型构建,在高盐(8%氯化钠)和普食(0.4%氯化钠)喂养条件下,观察小鼠尿钠排泄的变化、脂联素的分泌及达格列净(特异性SGLT2抑制剂)干预后肾脏SGLT2表达的变化。3.Adn-/-小鼠注射外源性脂联素Acrp30,观察其迁移表达特性。4.Adn+/+和Adn-/-小鼠分组培养后,予以PPARδ激动剂,免疫印迹法检测肾周脂肪及肾脏皮质SGLT2、脂联素的表达,观察小鼠尿钠、尿糖排泄及糖耐量变化。5.高盐及普食喂养db/db糖尿病小鼠后,观察小鼠尿钠、尿糖排泄等改变,予以达格列净干预,检测小鼠肾脏SGLT2的表达,并观察小鼠24小时尿钠、尿糖排泄及血糖变化。6.收集糖尿病患者血浆样本,检测患者空腹血糖、糖化血红蛋白、血清脂联素、24小时尿钠,分析各指标之间的相关性。结果:1.高盐(8%盐浓度)培养下3T3-L1前脂肪细胞和C57BL/6小鼠原代脂肪细胞中PPARδ表达显著升高,氯化钠和甘露醇(渗透压对照)以浓度依赖的方式显著增加PPARδ的表达。2.与Fabp4-PPARδflox/flox小鼠相比,8%高盐摄入明显降低PPARδflox/flox小鼠的空腹血糖水平。高盐摄入显著增加Fabp4-PPARδflox/flox小鼠和PPARδflox/flox小鼠尿量和尿钠排泄以及血浆和肾周脂肪的钠含量,同时降低血浆醛固酮水平。高盐摄入显著降低PPARδflox/flox小鼠肾脏皮质SGLT2表达。达格列净显著增加高盐喂养下PPARδflox/flox小鼠尿钠及尿糖排泄。3.高盐摄入的PPARδflox/flox小鼠肾周脂肪和肾皮质中脂联素表达显著增加,血浆脂联素水平在PPARδflox/flox小鼠升高而在Fabp4-PPARδflox/flox小鼠中降低。予以PPARδ激动剂GW501516处理PPARδflox/flox小鼠原代培养的脂肪细胞致脂联素表达升高,而PPARδ拮抗剂GSK0660则降低其表达。4.染色质免疫共沉淀的结果表明,脂联素(Acrp30)干预后,SLC5A2基因启动子部位HNF-1a和sp-1的结合水平显著降低。5.GW501516显著提高Adn+/+小鼠血浆脂联素水平和肾周脂肪、肾皮质脂联素的表达,但对Adn-/-小鼠无此效应,口服葡萄糖灌胃后,GW501516促进尿钠和尿糖排泄的作用在Adn-/-小鼠中削弱了。GW501516干预后肾脏SGLT2的表达明显增强,而高盐对肾脏SGLT2表达的抑制作用在Adn-/-小鼠中消除了。6.高盐饮食不仅增加db/db小鼠的尿钠排泄,也增加尿量和尿糖。db/db小鼠肾脏SGLT2表达显著高于db/m小鼠,而高盐摄入可下调其表达。达格列净干预后,高盐喂养的db/db小鼠尿钠排泄和尿糖的升高幅度低于db/m小鼠。7.在糖尿病患者中,尿钠排泄与空腹血糖、糖化血红蛋白存在显著的负相关关系。此外,血糖或糖化血红蛋白控制良好的糖尿病患者的尿钠排泄更高,尿钠排泄量和血浆脂联素水平之间有明显的相关性。结论:1.脂肪组织PPARδ激活促进小鼠在高盐摄入条件下的尿钠和尿糖排泄增加,这与肾脏近端小管上SGLT2表达及功能抑制有关。脂肪组织PPARδ介导的脂联素在抑制肾脏SGLT2中起着至关重要的作用。2.在生理情况下,高盐摄入诱导的糖尿病小鼠尿钠排泄受损源于高糖导致的SGLT2活性增加。3.血糖控制不佳的2型糖尿病患者尿钠排泄减少,且糖尿病患者的血浆脂联素水平尿钠排泄密切相关。
[Abstract]:Background and purpose: the management of hypertension in diabetic patients is the key to reducing cardiovascular mortality and morbidity. For a long time, the diet regulation scheme of hypertension focuses on salt intake. Therefore, the major guidelines recommend diabetes and hypertension patients to limit salt intake. However, the potential benefits of salt restriction are still controversial, some evidence is still disputed. It is shown that salt restriction increases cardiovascular and total mortality in patients with diabetes and hypertension, and the effect of high sugar diet on high blood pressure is far greater than high salt intake. However, there is still a lack of research on the close link between salt intake and blood glucose homeostasis. At the same time, it plays a vital role in the filtration and reabsorption of glucose. Among them, the sodium glucose co transporter (SGLTs), distributed on the side of the proximal tubular epithelial cell of the renal cortex, has the function of regulating sodium and sugar reabsorption, and can transport Na+ and glucose at high efficiency with 1:1. In high glucose state, the activity of SGLT2 Increased heavy absorption of glucose and sodium. In the diabetic rat model, SGLT2 mutation prevents high glucose induced multiple organ dysfunction. Selective SGLT2 inhibitors can improve hyperglycemia in diabetic patients and have a slight hypotensive effect. In the last 10 years, the peroxisome proliferator activated receptor (peroxisome P) Roliferator-activated receptor, PPAR) has become a new and effective target for the treatment of cardiovascular metabolic diseases. Human kidneys express three different PPAR subtypes, PPAR alpha, PPAR gamma and PPAR Delta, which play an important role in the body's glycolipid metabolism. Nowadays, the thiazolidane two ketone (TZD) drug is used for the treatment of diabetes by activating the fat tissue PP AR gamma improves insulin resistance and thus plays a role in improving blood sugar. However, studies have shown that TZD drugs may lead to sodium retention and heart failure, which greatly restricts the clinical application of.PPAR Delta expression in almost all cells and tissues, and is considered to be involved in the pathogenesis of many chronic diseases, including hypertension, diabetes, and atheromatous disease. It can effectively alleviate hyperlipidemia, blood glucose and insulin resistance in obese and diabetic rodents. In diabetic rats, specific excitated PPAR Delta expression shows renal protection in diabetic rats. In addition, the expression of fat adiponectin, which is increased by PPAR delta irritation agent, may be in the heart. However, it is not clear whether PPAR delta is involved in the treatment of sodium and transshipment of glucose by regulating adiponectin expression. Adiponectin is the most abundant protein product secreted by adiponectin, especially visceral adipose tissue. Adiponectin, through its specific receptors, AdipoR1 and AdipoR2, is in a variety of ways. It has been found that PPARs plays an important role in the regulation of the expression of adiponectin. The application of PPAR delta activator can significantly increase the level of visceral adiponectin, thus exerting a comprehensive effect on the improvement of cardiovascular metabolic disorders. Low level of adiponectin may be an important factor in the development of insulin resistance. Salt intake plays an important role in adiponectin secretion. Low salt intervention can significantly increase adiponectin secretion, and improve insulin resistance and systemic inflammatory response. However, how lipoplin participates in the metabolism of salt is not clear. We suggest that PPAR delta participates in the process of sodium transport and glucose reabsorption in renal tubule SGLT2 by regulating adiponectin expression, and interferes with the scientific hypothesis that one or more key targets in this link can maintain or restore sodium and glucose homeostasis, and carry out a series of cell, animal experiments and clinical validation, in order to verify the above It is assumed that this study is divided into two parts: 1, to verify the effect of high salt intake on blood glucose homeostasis and salt excretion, and to observe the effect of PPAR delta mediated adiponectin secretion on SGLT2 and its effect on urine sodium and urine sugar excretion and its mechanism.2. In diabetic animal model, the effect of high salt intake on SGLT2 and its mechanism are observed and in clinical practice. The results showed that the fat PPAR delta plays a key role in the adiponectin mediated SGLT2 regulating sodium glucose balance in the kidney. Materials and methods: This study includes two parts: 1. the effect of high salt on urinary sodium excretion and glucose homeostasis, and the promotion of kidney urine induced by adiponectin in mice after high salt induced adipose PPAR delta activation The molecular mechanism of sodium and glucose excretion, and testing the acute functional change of SGLT2 in the mouse model.2. on the basis of the first part of the experiment, the effect of high salt intake on SGLT2 expression and function in the db/db diabetic animal model was studied, and the relationship between blood sugar, adiponectin, and urine sodium three was analyzed in the clinical diabetic patients with.1.3T3-L1 pre fat. The fat cells and the primary adipocytes of C57BL/6 mice were grouped, and the expression of PPAR subtypes was detected by Western blot, and the osmotic pressure of sodium chloride and mannitol gradient was set up. As a control observation, the expression of PPAR delta was observed by.2. fat specific knockout PPAR Delta (Fabp4-PPAR Delta Flox /flox) model, in high salt (8% sodium chloride) and universal food (0 The changes of urine sodium excretion, secretion of adiponectin and the changes of SGLT2 expression in renal SGLT2 after intervention were observed in mice. The.3.Adn-/- mice were injected with exogenous adiponectin Acrp30 after intervention. The migration expression characteristics of.4.Adn+/+ and Adn-/- mice were observed, and PPAR delta agonists were immunized and immunized with PPAR delta activator. The expression of SGLT2, renal cortex and renal cortex, the expression of adiponectin, mice urine sodium, urine sugar excretion and glucose tolerance change.5. high salt and universal diet feeding db/db diabetic mice were observed. The changes of urine sodium and urine sugar excretion were observed in mice. The expression of SGLT2 in kidney of rats was measured and the expression of renal SGLT2 was detected in mice, and the urine sodium and urine sugar were observed for 24 hours in mice. Excretory and blood glucose changes.6. collected plasma samples of diabetic patients, tested the patient's fasting blood glucose, glycated hemoglobin, serum adiponectin, 24 hour urine sodium, and analyzed the correlation between the indexes. Results: the expression of PPAR Delta in 3T3-L1 preadipocytes and C57BL/6 rat primary adipocytes increased significantly under the 1. high salt (8% salt concentration) culture, and the sodium chloride and sodium chloride were significantly increased. Mannitol (osmotic control) significantly increased the expression of PPAR Delta.2. in a concentration dependent manner compared with Fabp4-PPAR Delta flox/flox mice. 8% high salt intake significantly reduced the fasting blood glucose level of PPAR Delta flox/flox mice. High salt intake significantly increased the urine volume and sodium excretion of PPAR Delta flox/flox mice and PPAR Delta flox/flox mice, as well as the plasma and plasma levels. Sodium content of perirenal fat and plasma aldosterone levels decreased plasma aldosterone levels. High salt intake significantly reduced the expression of SGLT2 in renal cortex of PPAR Delta flox/flox mice. Da glitjing significantly increased the expression of PPAR Delta flox/flox mice in the urinary sodium and urine sugar excretion of.3. high salt intake of PPAR Delta flox/flox mice, and the expression of adiponectin in the renal cortex and renal cortex was significantly increased. Adding, plasma adiponectin levels were increased in PPAR Delta flox/flox mice and decreased in Fabp4-PPAR Delta flox/flox mice. PPAR delta agonist GW501516 treated the primary cultured adipocytes of PPAR Delta flox/flox mice to increase the expression of adiponectin, while PPAR delta antagonist GSK0660 decreased its expression of.4. chromatin immunoprecipitation. After Acrp30, the binding level of HNF-1a and SP-1 at the promoter of SLC5A2 gene significantly decreased.5.GW501516 significantly increased plasma adiponectin level and perirenal adiponectin and renal cortex adiponectin expression in Adn+/+ mice, but had no effect on Adn-/- mice. After oral glucose administration, GW501516 promoted urine sodium and urine glucose excretion in Adn-/-. In mice, the expression of kidney SGLT2 was markedly enhanced after.GW501516 drying, and the inhibition of SGLT2 expression in the kidney was eliminated in Adn-/- mice by the inhibition of.6. high salt diet not only to increase the urine sodium excretion of db/db mice but also to increase urine volume and urine sugar.Db/db mouse kidney SGLT2 expression to be higher than db/m mice, while high salt intake could reduce the expression of SGLT2. The urine sodium excretion and urine sugar in db/db mice fed with high salt were lower than that of db/m mice.7. in diabetic patients. There was a significant negative correlation between sodium excretion and fasting blood glucose and glycosylated hemoglobin. In addition, the urine sodium excretion of diabetic patients with good control of glycemic hemoglobin was higher, and urine excretion was higher. There is a significant correlation between sodium excretion and plasma adiponectin levels. Conclusion: 1. PPAR delta activation in adipose tissue increases the excretion of urine and urine sugar in mice under high salt intake, which is related to the expression of SGLT2 and function inhibition on the proximal tubules of the kidney. Adipose tissue PPAR delta mediated adiponectin plays a critical role in inhibiting renal SGLT2. The role of.2. in physiological conditions is that the impaired urinary sodium excretion in diabetic mice induced by high salt intake is derived from high glucose induced SGLT2 activity and the decrease of urine sodium excretion in type 2 diabetic patients with poor control of.3. glucose control, and the plasma adiponectin level in diabetic patients is closely related to the urinary sodium excretion.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.1;R544.1

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